Figure 7

Histological analysis, cellular proliferation and apoptosis of regenerating limbs transfected with axolotl BMP-2 , Xenopus laevis Noggin and mRFP. Ectopic expression of axolotl BMP-2 (A-C), Xenopus laevis Noggin (D-F) and mRFP (G-I) by electroporating the expression plasmids directly into regenerating limbs were further analysed at the cellular level. Panels A, D and G show the resulting phenotypes at the tissue level on 10 μm sections with the Masson's trichrome staining. Clear differences can be observed between the ectopic expression of axolotl BMP-2 (A: condensation area pointed out by the black arrow), Xenopus laevis Noggin (D: no condensation visible) and the mRFP control (G, normal regenerate). Panels B, E and H show the level of BrdU incorporation as a measure of cell division. Overexpression of BMP-2 6 days after electroporation (B) did not seem to affect cell division compared to mRFP control (H). However, Noggin overexpression significantly reduced the number of BrdU positive cells 6 days after electroporation, especially on the posterior side where it was overexpressed (E). Panels C, F and I show the level of apoptosis as determined by TUNEL assay. BMP-2 overexpression (C) significantly increased the number of apoptotic cells during regeneration compared to Noggin (F), which reduced apoptosis, and mRFP control (I).